The Latin American Pesticide Residue Workshop (LAPRW) is a biannual open forum for discussion on current concepts and future developments in the field of pesticide residues in food and the environment. Pickering Laboratories was proud to support this workshop by sponsoring and also presenting a poster titled “Glyphosate in soy beans, corn and sunflower seeds by HPLC with post-column Derivatization and fluorescence detection”

Downtown Montevideo

If you would like a copy of the poster, please email support@pickeringlabs.com

Chromatography Quiz #3Identify the error made when running the Glyphosate chromatogram below and win a prize! Simply email your answer and your full contact information to Rebecca at rlsmith@pickeringlabs.com by July 1st in order to win. You will receive email confirmation when your submission is received, and the troubleshooting answer and winner congratulations will be published in the next issue (to be anonymous, please notify Rebecca in submission).

Baseline noise is a common and often frustrating problem in HPLC analysis. It makes integration difficult and adversely affects reproducibility and sensitivity of analysis. The most common sources of baseline noise are from pumps and bubbles in the lines or detectors. Not all noisy baselines can be easily explained but understanding common noise patterns will help you to determine what part of your system has a problem.

The Sine WaveTo observe the noise pattern you need to find a portion of the chromatogram that does not have any peaks and zoom in to look at about 5-10 min of the baseline. Baseline noise caused by reciprocating HPLC or post-column pump looks like a fairly regular sine wave. This kind of noise is usually due to old/poorly installed seals or bad piston. Ups and downs in the baseline follow flow/pressure variations as the piston moves. The period of the sine wave is different for different pumps. Measure the interval (in seconds) between the maximums of two waves to determine which pump is causing the noise. Most HPLC pumps have an interval of 6 -13 seconds. Pickering PCX5200 and Vector PCX reagent pumps have 2 sec and 4 sec intervals respectively. Pinnacle PCX contains a syringe pump which moves the piston in a single stroke hence it does not produce sine wave noise.

Most HPLC software programs record the column pressure during the analysis. It is very helpful to look at the trace to check if pressure variations have a similar pattern to your baseline noise. Pinnacle PCX users can also take advantage of log files collected by the Pickering software. Reagent pump pressure recorded in the log file helps Pickering technical support to evaluate the performance of the post-column system and determine if the syringe pump needs maintenance.

BubblesBaseline noise caused by bubbles consists of random spikes of varying amplitudes. Bubbles can occur in solvent lines or in the detector flow cell and are often caused by solutions outgassing. To prevent this from happening use a properly working degasser and install a backpressure regulator on the detector waste line to prevent boiling and outgassing in the heated reactor.

Detector NoiseDetector noise is always present and can be visible even on a “good” baseline if you zoom in deep enough. It is random and looks about the same throughout the chromatogram. An old detector lamp, dirt in a flow cell or problems with electronics can greatly increase noise level. If detector noise is suspected make sure the flow cell is clean and check the lamp hours. Built-in detector tests are also useful in assessing detector performance.

Shooting in the DarkA common mistake people make when troubleshooting baseline noise in post-column applications is turning off the post-column reagent pump. Noise in the baseline is essentially variations in signal so it is proportional to background signal. Common eluants don’t fluoresce or absorb light in the visible range so when eluants alone go through the detector there is no signal and hence no noise. Post-column reagents, on the other hand, are often either colored or have background fluorescence so elevated noise caused by any part of HPLC system becomes visible. Turning off the reagent pump is akin to turning off the detector lamp and taking a shot in the dark – the noise is still there but we just can’t see it.

Let Us HelpWhen contacting Pickering support about elevated baseline noise please be ready to fax or e-mail your chromatogram and zoomed in portion of the baseline. For Pinnacle PCX users sending the log files will also help us to find the problem. You can email support@pickeringlabs.com or send a fax to 650-968-0749.

RI sensitivity applies only to UV-vis detectors. There are two sources of RI noise in post-column applications:

– RI discontinuities due to imperfect mixing.– RI discontinuities due to temperature gradients in the eluant/reagent stream as it leaves a heated reactor.

In either case, when such inhomogeneities enter the flowcell, they bend light into the wall or off the photomultiplier tube, causing detector noise. The noise usually correlates with the piston cycles of the pumps, thus limiting the detector to low-sensitivity applications.Most flowcells in modern UV-vis detectors are designed to minimize the effects of RI.

In order to minimize the temperature-related RI effects mentioned above, some manufacturers have a capillary heat exchanger at the flowcell entrance. In some instances this heat exchanger has an internal diameter of 0.12 mm (0.005 inches), which can result in post-column pressures in excess of 42 bar (600 psi). Since this can exceed the pressure rating of a heated reactor made with fluorocarbon tubing, this small-diameter heat-exchanger tube may need to be replaced with a 0.25 mm (0.010 inch) i.d. tube.

Detector Pressure Ratings

When the eluant-reagent stream from the heated reactor reaches the detector, it can release dissolved gas as it cools. The Pickering Laboratories derivatization instruments place a back-pressure of 7 bar (100 psi) on the detector flowcell in order to prevent the formation of bubbles.

– suppress boiling in the reactor– prevent outgassing in the detector flowcell.

The back-pressure regulator can be factory-adjusted to accommodate flowcells with a lower back-pressure rating, depending upon the reactor temperature. but a setting lower than 3.2 5 bar (70 psi) is not recommended for reactor temperatures over 100° C.

Operating an HPLC system with a post-column derivatization system can be as routine as regular LC. The benefits from this LC/post-column combination include minimal sample pre-treatment, greatly improved sensitivity, and enhanced selectivity for compounds that would normally be much more difficult to detect.

Stability of Reagent: The minimum reagent stability sufficient for routine work is one day. This means that the yield and signal-to-noise ratio for a given sample must remain constant for at least 8 hours.

Completeness of Reaction: The analytical separation is complete when the reagent is mixed with the column effluent. Therefore, in order to minimize band spreading, it is important to keep the volume small between the mixing tee and the detector. If the reaction is slow (in excess of one minute), an elevated temperature can be used to decrease the reaction time.

Reproducibility: Unless the system consistently produces the same signal for the same sample, quantitation is impossible. Because the reaction is occurring ³on the fly² as the combined column and reagent stream flows toward the detector, the reproducibility is linked to the flow-rate precision of the pumps and to the temperature. Accordingly, even an incomplete reaction will be as repeatable as the retention time for any given species. The completeness of the reaction, then, is not strictly necessary for reproducibility, but it is important for maximum sensitivity.

Minimal Detector Response of Reagents: The color or background fluorescence of the reagent (or its by-products) represents a continuous noise source. Because the reagent is present in excess relative to the analyte, the analyte’s signal could be obliterated by the reagent’s strong background signal. Pickering’s Chromatographic Grade® eluants and reagents are guaranteed to produce the absolute minimum possible detector background signal in post-column applications.

Solubility: All species must remain in solution, including the combined components of the eluants and the reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors and foul detector flowcells.

Ninhydrin chemistry provides a good example of multiple solubility considerations. Ninhydrin reagents contain a lithium acetate buffer, ninhydrin, hydrindantin, and a water-miscible organic solvent. The organic solvent is necessary to maintain both the hydrindantin and the new purple chromophore (derivative) in solution. Also the presence of lithium ion in the formula precludes the use of eluants containing phosphate, because lithium phosphate is insoluble and would precipitate at the point of mixing.

Chromatography is a science of separations. High Performance Liquid Chromatography (HPLC) like other forms of chromatography, is used to separate complex mixtures into their components. There are many flavors of HPLC, but what they have in common is that the separation takes place in solution. Having separated a mixture, you need to see the components. The most popular detectors use either UV/VIS light absorption, or fluorescence. Unfortunately, many substances are difficult to detect. Moreover, you want to see the components of interest without distraction from the background.

Post-column derivatization, also known as post-column reaction, renders visible certain compounds that are normally invisible. This trick is accomplished after the separation by performing a chemical reaction on the substances that gives them an easily-detectable physical property. Typically you use a reaction that produces a strong color or makes a fluorescent product. You can increase the sensitivity of detection by several orders of magnitude in favorable cases. Most reagents are selective for a particular class of substances, so analytes of that class are more easily seen against a complex background. So, post-column derivatization is used to increase sensitivity and selectivity in HPLC analysis.

The post-column reaction system mixes the stream of eluant from the HPLC column with a stream of reagent solution. The mixture usually flows through a reactor to allow enough time for the chemical reactions to complete. If the reaction is slow, the reactor may be heated to speed things up. Some reactions need two or more reagents added in sequence. Finally the mixed streams pass into the detector, typically UV/VIS absorbance or fluorescence. Of course a practical system requires metering pumps, pulse-dampeners, thermostats, and safety systems to give reliable results.

Examples of the chemistry and hardware are given in the catalog and user’s manuals.

As of right now, Pickering Laboratories, Inc. will remain open during the “Shelter in Place” order for Santa Clara County so as to provide other essential businesses with the support and supplies necessary to operate, including hospitals and clinical laboratories.

Our inventory levels and response times are currently normal. Responsiveness is subject to change and orders may need to be prioritized accordingly to supply essential businesses.

Thank you for your patience and for your business as we all work together through this difficult time. Please contact us with any questions you may have.